Cryptococcus neoformans is an opportunistic fungal pathogen in patients with AIDS. C. neoformans is a basidiomycetes widely diverged from most other fungal pathogens. The most common clinical presentations are meningoencephalitis and pulmonary cryptococcosis. Available antifungal agents, directed against the ergosterol pathway, are inadequate for continued safe and effective therapy due to inherent toxicity, emerging fungal resistance, and the requirement for lifelong treatment. New targets for antifungal therapies are needed. Proteins that are differentially regulated during infection may be essential for pathogenesis and be good targets for novel antifungal therapies. Over the past decade, the pathogenesis of C. neoformans has been intensively studied using molecular biology, genetic, immunological and biochemical approaches. However, only a handful of new antifungal targets have been identified using traditional methods. A funded genome project for C. neoformans has made new approaches possible. We have developed a proteomic system for C. neoformans that utilizes 2-dimensional gel electrophoresis and MALDI-TOF analysis. We will reproducibly separate and quantitate proteins, identify proteins that are affected by specific conditions, and determine the identity of those proteins. We propose to use proteomics to identify C. neoformans proteins that are affected by specific in vitro conditions that mimic an aspect of the infection process. These proteins will form the basis for comparison for additional studies examining proteins that are affected in an animal model. The genes encoding these proteins will be mutated by targeted gene disruption and tested for their role in viability and pathogenesis using a mouse model.